Screening the methanol extracts of some Iranian plants for acetylcholinesterase inhibitory activity

Acetylcholinesterase (AChE) is the main enzyme for the breakdown of acetylcholine. Nowadays, usage of the inhibitors of this enzyme is one of the most important types of treatment of mild to moderate neurodegenerative diseases such as Alzheimer’s disease. Herbal medicines can be a new source of inhibitors of this enzyme. In this study we examined around 100 different plants to evaluate their inhibitory properties for AChE enzyme. Plants were scientifically identified and their extracts were prepared by methanol percolation. Acetylcholinesterase activity was measured using a colorimetric method in the presence or absence of the extracts. Eserine was used as a positive control. Methanol extracts of the Levisticum officinale, Bergeris integrima and Rheum ribes showed more than 50% AChE inhibitory activity. The inhibition kinetics were studied in the presence of the most effective extracts. L. officinale and B. integrima inhibited AChE activity in a non-competitive manner, while R. ribes competitively inhibitied the enzyme as revealed by double-reciprocal Linweaver-Burk plot analysis. Under controlled condition, Km and Vmax values of the enzyme were found to be 9.4 mM and 0.238 mM/min, respectively. However, in the presence of L. officinale, B. integrima, and R. ribes extracts, Vmax values were 0.192, 0.074 and 0.238 mM/min, respectively. Due to the competitive inhibition of the enzyme by R. ribes extract, the Km value of 21.2 mM was obtained. The concentration required for 50% enzyme inhibition (IC50 value) was 0.5, 0.9, and 0.95 mg/ml for the L. officinale, B. integrima and R. ribes extracts, respectively. The IC50 of the eserine was determined to be 0.8 mg/ml.


INTRODUCTION
The main role of acethylcholinestrase (AChE) is to rapidly hydrolyze acetylcholine at the cholinergic synapses, ending the transmission of nerve impulses (1).The use of AChE inhibitors in order to enhance cholinergic function in the brain is the main strategy in treatment of Alzheimer's disease (AD) which is characterized by loss or decline in memory and cognitive impairment (2,3).AD is the most common cause of dementia in the elderly and is responsible for 60 to 80 percent of the cases (2).Degeneration and loss of basal forebrain cholinergic innervation is accepted as a major cause of cognitive impairment and memory loss for the disease (4)(5)(6).
The pathological hallmarks of AD are the senile plaques and neurofibrillary tangles.The accumulation of plaques and tangles, and the progression of other pathological processes, leads to a massive neuronal loss, which is usually preceded by synapse loss (1).Several AChE inhibitors such as tacrine, donepzil, rivastigmine and galanthamine, are available for the treatment of mild to moderate AD (7).Although the use of these drugs are beneficial in the treatment of AD symptoms, they may also cause some adverse side effects (8).The most common side effects of these drugs include: anorexia, diarrhea, fatigue, nausea, muscle cramps as well as gastrointestinal, cardiorespiratory, genitourinary and sleep disturbances (6,8).Therefore, cheaper and 106 safer AChE inhibitors with higher efficacy, bioavailability and fewer side effects, particularly from natural sources, have been extensively investigated and research should be continued.

Plant material
Different parts of all plants such as flowers, fruits, seeds, aerial parts and roots were collected from various provinces of Iran or obtained from the medical herbal markets in Kerman city.Scientific names of the collected plants were authenticated.A voucher specimen of each plant was retained in the herbarium in the Herbal Medicine Research Center, Faculty of the Pharmacy, Kerman University of Medical Sciences, Kerman, Iran.

Extraction of plant material
Each plant was air dried in the dark, and grounded into fine powder.The powdered materials (20 g) were extracted with 200 ml of absolute methanol for 24 h at room temperature.The suspensions were then filtered and air-dried.The extracts were stored at -20 °C until being used (9).

Acetylcholinesterase activity assay
The AChE activity was measured according to the method developed by Ellman et al. (10).This method employs ATCI as a synthetic substrate for AChE.In this procedure 10 µl of methanol extract containing 50 µg of crude extract was added to the reaction mixture containing 20 µl of enzyme solution (0.1 U/ml) and 950 µl sodium phosphate buffer (pH 8, 0.1 M ).Reaction mixture was incubated for 15 min at 25 ºC.Then, 10 µl of DTNB (10 mM) was added and the reaction initiated by the addition of substrate (10 µl of ATCI 14 mM solution).Based on this procedure, ATCI is broken down to thiocholine and acetate by the enzyme and thiocholine is reacted with DTNB to produce yellow color.The intensity of yellow color was measured at 410 nm after 10 min.Eserine (20 µl of 0.1 mM solution in phosphate buffer) was used as positive control.The percentage of enzyme inhibition was calculated using the following formula (11).Inhibition% = 100 -[A t /A c × 100] where, A t is the absorbance of the tested extract and A c is the absorbance of the standard control.

Kinetic study
In order to elucidate the type of inhibition of the effective extracts, the enzyme activity was measured in the presence of an increasing concentrations of ACTI (2-20 mM), and in the presence or absence of two concentrations of each extract (4 and 8 mg/ml).Inhibition mode was determined by double-reciprocal Lineweaver-Burk plot analysis of the data resulted from enzyme assays containing various concentrations of ACTI and the extracts.

Plants with acetylcholinesterase inhibitory effect
Among all plants examined, B. integrima, L. officinale and R. ribes showed the strongest inhibition on the enzyme activity (80.2, 97.6 and 72.4%, respectively).Alhagi camelorum, Marrubium anisodon, Vaccinium arctostaphilus, Peganum harmala, Rosa damascene and Valeriana hispida, Myrtus communis, Nepta saccharata and Quercus infectoria showed inhibitory effect between 20-50%.The other plants showed inhibitory effect less than 20% or had no effect on enzyme activity.Acetylcholinesterase inhibitory activity of all plants is shown in Table 1.

Kinetic analysis
The inhibition modes of the three most active plant extracts were analyzed by doublereciprocal Lineweaver-Burk plot.B. integrima and L. officinale inhibited the enzyme activity in a non-competitive manner (Fig. 1 and 2), whereas R. ribes showed competitive inhibition (Fig. 3).The K m value of the    ribes were 0.5, 0.9, and 0.95 mg/ml, respectively (Table 2).The K i values of 1.6, 5.5 and 6.37 mg/ml were found for L. officinale, B. integrima and R. ribes, respectively.

DISCUSSION
Acetylcholinesterase inhibitors are used for the treatment of Alzheimer's disease.These inhibitors may interact with the central cholinergic system function to improve memory and cognitive disorders in the patients by decreasing the breakdown of acetylcholine in brain synapses (12).Nature is an unlimited resource for providing chemicals and biological compounds which are unique and complex insofar as their chemical synthesis seems impossible.The anti-cholinesterase activity of some plants in the world has been approved (12).In this study we concluded that roots of L. officinale and B. integrima and rhizomes of R. ribes possess a strong anticholinesterase activity.IC50 values (concentration required to inhibit 50% of enzyme activity) were calculated from the regression equation obtained from various concentrations of the test compounds (Table 2).Previously, it has been demonstrated (13) that the methanol extracts of punica granatum and Rosa damascene have more than 50% inhibitory effect on alpha manosidase activity, but these extracts did not show strong inhibitory effects on acetylcholinesterase. Extract of L. officinale exhibited strong inhibitory effects on the alpha glucosidase and the pancreatic lipase but exhibited weak inhibition on alpha manosidase (13,14).The plant extract demonstrated apoptotic activity on humans leukaemia cell line (15), and had an anti-mycobacterial activity as well (16).B. integrima produced no effect on the alpha glucosidase and the pancreatic lipase activity (14,17).1methylmalate, one of the B. integrima fruit components, increased the anti-microbial activity against Staphylococcus aureus (18).Isoquinoline alkaloids attained from the root of Turkish berberis species showed an antiinflamatory and anti-nociceptive effects (19), whereas in our study this plant exhibited the anti-cholinesterase activity.R. ribes has shown to have hypo-glycemic effects in the alloxan induced diabetic rats but did not reveal hypoglycemic activity in healthy mice (20).It was also demonstrated that the herb possesses some anti-depressive activity (21).Some drugs such as rasagiline, used in the treatment of Alzheimer's disease, retain the neuroprotective properties with their anti-cholinesterase and monoamine oxidase inhibitory effects and, has shown anti-depressant activity in animals (22).Therefore, the anti-depressant properties of R. ribes could be due to the anti-cholinesterase activity shown in this study.Other findings indicated that some components of R. ribes extract demonstrated selective cytotoxic activity on cancerous cell lines (23).The stem and root of R. ribes exhibited an antioxidant activity (24), but this plant did not have any inhibitory effect on the alpha glucosidase or the pancreatic lipase activity (14,17).The methanol extracts of Rosa damascene, Quercus infectoria, Eucalyptus galbie, Myrtus communis, Terminalia chebulla, Punica granatum, Camellia sinensis, and Cinnamomum zeylanicum showed more than 50 percent inhibitory activity on the pancreatic lipase or alpha glucosidase (14,17), while they revealed no or little effect on cholinesterase activity.One of the most important anticholinesterase drugs, tacrine, proved to have both competitive and non-competitive inhibitory activities on acetylcholinesterase (25).Tolserin, the novel experimental AD therapeutic agent, inhibits the acetylcholinesterase in a non-competitive manner (26).R. ribes showed some similarities in kinetic properties to tacrine.L. officinale and B. integrima were non-competitive inhibitors of the enzyme, as acting similar to tolserin.A competitiveinhibitor binds to the active site of the enzyme and affects K m of the reaction.Components of R. ribes extract with competitive inhibition, may bind to the enzyme and block its activity.
When an inhibitor binds to the enzyme and/or enzyme-substrate complex, it is considered as non-competitive inhibition where the inhibitor affects only the V max of the reaction but has no effect on complex formation between the enzyme and the substrate.Therefore, two extracts that showed non-competitive inhibition on activity, probably have components that bind to enzyme or enzyme-substrate complex (27).

CONCLUSION
According to our results, it is possible to assume that R. ribes may contain some components that are functionally or structurally similar to tacrine.The same might be true for L. officinale and B. integrima regarding the kinetic properties of the tolserin.Results of this study indicated that these plants may offer great potentials for the treatment of different diseases including AD, and their antiacetylcholinesterase properties introduce them as promising candidates for more detailed in vitro and in vivo studies.Besides, these plants can be examined in order to isolate and identify the active ingredients, and this may serve as a foundation to find safer and more effective agent (s) for therapeutic use.

Fig. 1 .
Fig. 1.The Lineweaver-Burk plot of kinetic analysis of acetylcholinestrase at two different concentrations of L. officinale (4 and 8 mg/ml) in the presence of four different ATCI concentrations.

Fig. 2 .
Fig. 2. The Lineweaver-Burk plot of kinetic analysis of acetylcholinestrase at two different concentrations of B. integrima (4 and 8 mg/ml) in the presence of four different triolein concentrations.

Fig. 3 .
Fig. 3.The Lineweaver-Burk plot of kinetic analysis of acetylcholinestrase at two different concentrations of R. ribes (4 and 8 mg/ml) in the presence of four different ATCI concentrations.

Table 1 .
Acetylcholinesterase inhibitory activity of plants.

Table 1 . (Continued) Plants name Family Used part Inhibition %
N.E: No Effect

Table 2 .
The IC50 values of the methanol extracts compared to eserine as a positive control.